Highly Dynamic and Sex-Specific Expression of microRNAs During Early ES Cell Differentiation

Abstract

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of the mammalian blastocyst. Cellular differentiation entails loss of pluripotency and gain of lineage-specific characteristics. However, the molecular controls that govern the differentiation process remain poorly understood. We have characterized small RNA expression profiles in differentiating ES cells as a model for early mammalian development. High-throughput 454 pyro-sequencing was performed on 19–30 nt RNAs isolated from undifferentiated male and female ES cells, as well as day 2 and 5 differentiating derivatives. A discrete subset of microRNAs (miRNAs) largely dominated the small RNA repertoire, and the dynamics of their accumulation could be readily used to discriminate pluripotency from early differentiation events. Unsupervised partitioning around meloids (PAM) analysis revealed that differentiating ES cell miRNAs can be divided into three expression clusters with highly contrasted accumulation patterns. PAM analysis afforded an unprecedented level of definition in the temporal fluctuations of individual members of several miRNA genomic clusters. Notably, this unravelled highly complex post-transcriptional regulations of the key pluripotency miR-290 locus, and helped identify miR-293 as a clear outlier within this cluster. Accordingly, the miR-293 seed sequence and its predicted cellular targets differed drastically from those of the other abundant cluster members, suggesting that previous conclusions drawn from whole miR-290 over-expression need to be reconsidered. Our analysis in ES cells also uncovered a striking male-specific enrichment of the miR-302 family, which share the same seed sequence with most miR-290 family members. Accordingly, a miR-302 representative was strongly enriched in embryonic germ cells derived from primordial germ cells of male but not female mouse embryos. Identifying the chromatin remodelling and E2F-dependent transcription repressors Ari4a and Arid4b as additional targets of miR-302 and miR-290 supports and possibly expands a model integrating possible overlapping functions of the two miRNA families in mouse cell totipotency during early development. This study demonstrates that small RNA sampling throughout early ES cell differentiation enables the definition of statistically significant expression patterns for most cellular miRNAs. We have further shown that the transience of some of these miRNA patterns provides highly discriminative markers of particular ES cell states during their differentiation, an approach that might be broadly applicable to the study of early mammalian development.

Author Summary

The discovery of the first microRNA (lin-4) in C. elegans in 1993 and the increasing realization that small RNAs are at the heart of many biological processes have led to a revolution in our thinking about development and disease. In animals, several hundred microRNAs (miRNAs) have been identified that regulate diverse biological processes ranging from cell metabolism to cell differentiation and growth, apoptosis, and cancer. Moreover, it has been shown that many miRNAs are characterized by highly specific spatial and temporal expression patterns supporting their role in such processes. However, the dynamics of small RNA patterns in male and female embryonic stem (ES) cells in the course of early differentiation has not been investigated so far. Our work represents the first study of this kind. Notably, we have identified new classes of miRNAs that show extremely defined temporal profiles during ES cell differentiation, as well as sex-specificity. Our results are of broad interest and importance because they raise the power of ES cells in defining the repertoire of small RNAs and their dynamics in mammals, and underline the importance of integrating miRNA expression patterns into the transcription factor networks and epigenomic maps defined in ES cells in order to provide a better understanding of the control of pluripotency and lineage commitment.